1. Field of the Invention
The present invention is in the field of crystalline forms of polypeptides corresponding to the kinase domain of spleen tyrosine kinase (SYK), methods of obtaining such crystals, and to methods for identifying pharmacophores from high-resolution X-ray diffraction structures. The invention is also related to the field of using three-dimensional coordinates for screening for, identifying, and/or discovering compounds useful as inhibitors of SYK activity.
2. Summary of the Related Art
Spleen tyrosine kinase (SYK) is a member of the SYK family of tyrosine protein kinases, a family of cytoplasmic tyrosine kinases characterized by the presence of two SH2 domains in the amino terminal of a single kinase domain. Homologs of the SYK family of protein kinases have been identified in a number of species, including the human ZAP-70. SYK has been reported to be involved in several cellular signaling events. For example, SYK participates in immunoreceptor signaling, integrin signaling, and G protein-coupled receptor signaling. SYK is known to be expressed in hematopoietic cells as wells as in fibroblasts, epithelial cells, hepatocytes, neuronal cells, endothelial cells and mast cells. SYK is also involved in hematopoietic responses such as proliferation, for example, SYK inhibitors have been suggested as modulators of thrombin-induced ASM cell proliferation, differentiation, and phagocytosis.
In addition, SYK inhibitors have also been demonstrated to be important in non-hematopoietic cells as well, such as in fibroblasts, epithelial cells, breast tissue, hepatocytes, neuronal cells, and vascular endothelial cells. Accordingly, SYK has been implicated as playing a critical role in endothelial cell functions, including morphogenesis cell growth, migration, and survival, and as contributing to maintaining vascular integrity in vivo. For further review, see Yanagi et al., Biochem. Biophys. Res. Comm. 288:495-498 (2001).
Both SYK antisense and specific inhibitors have been shown to have some activity in asthma models and SYK is thought to be a target for the treatment of asthma and other airway diseases, as well as for allergies, inflammation, and autoimmunity. SYK has also been suggested as a target for the development of agonists in cancer therapy, due to its role in cell growth.
Knowledge of the 3-D structures of target proteins provides an important basis for structure-based approaches to drug design by defining the topographies of the complementary surfaces of ligands and their protein targets. Therefore, knowledge of the structure of the SYK protein may be useful in the identification, design, or development of novel and specific modulators of SYK, as well as diagnostic and pharmaceutical compounds useful for disorders associated with SYK expression or activity. Knowledge of the structure of SYK may also be useful for gene therapy. The three-dimensional structure of SYK may be useful, for example, for identifying novel therapeutic compounds that can modulate protein kinase activity, and for treatment of conditions mediated by human signal transduction of kinase activity, such as cancer, allergy, inflammation, asthma, arthritis, irritated bowel syndrome, and multiple sclerosis.
The molecular structure coordinates of SYK has been previously reported. For example, WO 04/035604 describes the crystal structure of SYK with stable ATP surrogates, such as AMP-PNP. Further, the coordinates of SYK co-crystals with Gleevec and Staurosporine have also been reported (Atwell et al., J. Biol. Chem. 279 (53):55827-55832 (2004); PDB ID: 1XBB and 1XBC). However, the currently available three-dimensional structures of SYK with or without bound ligands are not sufficient for identifying pharmacophores. The availability of pharmacophore models are desirable to facilitate the identification of modulators of SYK activity.
Thus, the present invention provides methods for identifying pharmacophore models using co-crystals of SYK and its ligands, and methods for identifying compounds that inhibit SYK using the pharmacophore models.